Use of an epitope of vascular endothelial growth factor receptor KDR/Flk-1

ABSTRACT

The present invention relates to the use of the epitope which comprises the tyrosine at position 1214 in the amino acid sequence of the vascular endothelial growth factor receptor, KDR/Flk-1, as a marker in the measurement of a change in the activation state of the KDR/Flk-1 receptor and to probes, such as antibodies, which recognize said epitope. The invention also relates to the use of KDR/Flk-1 epitope Y1214 as a marker in the detection of and/or measurement of the level of the KDR/Flk-1 receptor and to assays which utilize the use of the Y1214 epitope and to compounds derived from said assays.

The present invention relates to the use of the Y1214 epitope of theKDR/Flk-1 receptor as a marker in the measurement of a change in theactivation state of the KDR/Flk-1 receptor. The invention also relatesto the use of the KDR/Flk-1 epitope Y1214 as a marker in the detectionof and/or measurement of the level of the KDR/Flk-1 receptor and toassays which utilise the use of the Y1214 epitope and to compoundsderived from said assays.

KDR/Flk-1 is one of the receptors which binds vascular endothelialgrowth factor (VEGF), a growth factor which is engaged in the processesof angiogenesis. Three receptors have been identified which bind VEGF,VEGFR-1 (also known as Flt-1), VEGFR-2 (also known as KDR or Flk-1) andVEGFR-3 (also known as Flt-4). For more information on the receptorsbound by VEGF the reader is referred to the following review articles:Clauss, 2000 Seminars in Thrombosis and Hemostasis 26: 561-569; McMahon2000, The Oncologist 5: 3-10; and Karkkainen & Petrova, 2000, Oncogene19: 5598-5605.

Normal angiogenesis plays an important role in a variety of processesincluding embryonic development, wound healing and several components offemale reproductive function. However, angiogenesis is also involved ina number of disease conditions. For example, undesirable or pathologicalangiogenesis has been associated with disease states including diabeticretinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi'ssarcoma and haemangioma (Fan et al, 1995, Trends Pharmacol. Sci. 16:57-66; Folkman, 1995, Nature Medicine 1: 27-31). VEGF also has effectson vascular permeability, which is thought to play a role in both normaland pathological physiological processes (Cullinan-Bove et al, 1993,Endocrinology 133: 829-837; Senger et al, 1993, Cancer and MetastasisReviews, 12: 303-324).

The observation that angiogenesis is involved in a number of diseaseconditions has led to a number of approaches being undertaken to treatdiseases by the modulation of angiogenesis. One of these approaches hasbeen to develop inhibitors of the activity of receptors which bind VEGF.VEGF receptors belong to a family of receptors, called receptor tyrosinekinases (RTKs). RTKs are important in the transmission of biochemicalsignals across the plasma membrane of cells. These transmembranemolecules characteristically consist of an extracellular ligand-bindingdomain connected through a segment in the plasma membrane to anintracellular tyrosine kinase domain. Binding of ligand to the receptorresults in stimulation of the receptor-associated tyrosine kinaseactivity which leads to phosphorylation of tyrosine residues on both thereceptor and other intracellular molecules. These changes in tyrosinephosphorylation initiate a signalling cascade leading to a variety ofcellular responses. (For a review of RTKs, the reader is referred to:Schlessinger, 2000, Cell, 103:211-255. Examples of inhibitors of theVEGF receptor tyrosine kinase (VEGF RTK) can be found in the followingpatent applications: International Application, Publication NumberWO97/42187 and International Application, Publication Number:WO98/13350.

In addition to the need for inhibitors of the VEGF RTK, there is also aneed for tools to characterise the activation and deactivation of theVEGF RTK. Such tools would be useful, for example, in studiescharacterising the biochemistry of the VEGF RTK and in the measurementof changes in the activation state of the VEGF RTK during clinicalstudies of inhibitors of the receptor, thus giving an indication of thetherapeutic efficacy of said inhibitors.

One approach to the development of such tools is to look for amino acidepitopes on the VEGF receptor which are phosphorylated during activationof the VEGF RTK. Such epitopes could be used, for example, in followingthe activation and de-activation states of the VEGF RTK Takahashi et al,2001, EMBO Journal 20: 2768-2778 have identified two epitopes on theKDR/Flk-1 sub-type of the VEGF receptor, these are the tyrosine residueslocated at positions 1175 and 1214 in the amino acid sequence of theKDR/FLk-1 receptor (referred to hereafter as Y1175 and Y1214respectively). In this paper the authors characterised these twoepitopes and observed that Y1175 but not Y1214 was a major phospholipaseC-gamma (PLCγ) binding site linked to activation of MAP kinase, animportant protein downstream of the biochemical pathways which isindirectly activated by binding of VEGF to the KDR/Flk-1 receptor. Thus,the authors concluded that Y1175 but not Y1214 plays a crucial role inthe transduction of signals from the KDR/Flk-1 receptor to the MAPkinase pathway and DNA synthesis in endothelial cells. Thus, teachingthat Y1175, but not Y1214, would be an important marker for followingactivation of the KDR/Flk-1 receptor. We have raised antibodies againstthe Y1214 epitope and surprisingly found that we could use this epitopeto follow activation of the KDR/Flk-1 receptor, observing inhibition ofthe phosphorylation of Y1214 by inhibitors of the KDR/Flk-1 RTK (asshown in FIG. 1 below). We have also found that inhibitors of theKDR/Flk-1 RTK also inhibit phosphorylation of MAP Kinase in parallelwith inhibition of the phosphorylation of the Y1214 epitope. Thus,surprisingly we have found that Y1214 is an important marker forfollowing the activation of the KDR/Flk-1 receptor.

Thus, according to the first aspect of the present invention there isprovided the use of the KDR/Flk-1 epitope Y1214 as a marker in themeasurement of a change in the activation state of the KDR/Flk-1receptor.

The Y1214 epitope could be used to detect the presence of the KDR/Flk-1receptor in biological samples and to measure the levels of theKDR/Flk-1 receptor in biological samples. Thus, according to a furtherfeature of the first aspect of the invention there is provided the useof the KDR/Flk-1 epitope Y1214 as a marker in the detection of and/ormeasurement of the level of the KDR/Flk-1 receptor.

One approach to allow such detection and/or measurement of the KDR/Flk-1receptor is to develop probes to the Y1214 epitope, these could bedeveloped to either the phosphorylated or de-phosphorylated epitope.Thus, according to a second aspect of the invention there is provided aprobe directed to the KDR/Flk-1 epitope Y1214. Examples of such probesinclude binding proteins such as antibodies.

According to a further feature of the second aspect of the inventionthere is provided an antibody which specifically binds to epitope Y1214.A peptide fragment of the KDR/Flk-1 receptor comprising the Y1214epitope may be utilized to prepare antibodies that specifically bind toY1214. A specific example of such antibody preparation is described inExample 1 herein.

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, a peptide comprising the Y1214 epitope is administered tothe host animal typically through parenteral injection. Theimmunogenicity of a peptide comprising the Y1214 epitope may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to the Y1214 epitope.Examples of various assays useful for such determination include thosedescribed in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), (1980), each of which isincorporated herein by reference.

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) each of whichis incorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394 (1989),which is incorporated herein by reference. Monoclonal antibodies canalso be produced using cell lines, such as CHO cells or NS0 cells, ormicro-organisms, such as bacteria or yeast, into which the genes for theheavy and light chains of the antibody have been transfected. Examplesof such methods can be found in U.S. Pat. Nos. 6,331,415, 5,876,961,5,545,403 and 5,807,715, each of which is incorporated by reference.

Thus, according to a further aspect of the invention there is provided amonoclonal antibody which specifically binds to the KDR/Flk-1 epitopeY1214.

According to a further aspect of the invention there is a provided ahybridoma which produces an antibody which specifically binds to theKDR/Flk-1 epitope Y1214, preferably a monoclonal antibody.

According to a further aspect of the invention there is provided arecombinant cell line transfected with the cDNA for the expression ofthe heavy and light chains for an antibody which specifically binds tothe KDR/Flk-1 epitope Y1214.

Once isolated and purified, the antibodies against Y1214 may be used todetect the presence of Y1214 in a sample using established assayprotocols. In addition such antibodies could be used to measure thelevels of the KDR/Flk-1 receptor in samples. Further, the antibodies ofthe invention may be used therapeutically to bind to the Y1214 epitopeand inhibit its activity in-vivo.

Thus, according to a further aspect of the invention there is providedthe use of an antibody which binds to the KDR/Flk-1 epitope Y1214 in themanufacture of a medicament, preferably a medicament for the treatmentof cancer.

According to a further aspect of the invention there is provided amethod of treatment using an antibody which binds to the KDR/Flk-1epitope Y1214, preferably a method for the treatment of cancer.

Another approach to the use of the KDR/Flk-1 epitope Y1214 as a markerin the measurement of a change in the activation state of the KDR/Flk-1receptor is to measure the phosphorylation state of the KDR/Flk-1receptor activated in the presence of adenosine triphosphate labelledwith radioactive phosphate (for example, labelled with phosphorous 32)and to determine the level of radioactivity of the KDR/Flk-1 receptor,preferably the level of radioactivity in peptide fragments of theKDR/Flk-1 receptor comprising Y1214.

A further approach to the use of the KDR/Flk-1 epitope Y1214 as a markerin the measurement of a change in the activation state of the KDR/Flk-1receptor is to use nuclear magnetic resonance (NMR) to follow changes inthe phosphorylation state of Y1214.

According to a further aspect of the invention there is provided amethod of generating a probe directed to the Y1214 epitope of theKDR/Flk-1 receptor. Preferably the generation of an antibody.

According to a further aspect of the invention there is provided amethod of generating an antibody, which comprises:

-   (i) immunizing a mammal with a peptide which comprises the KDR/Flk-1    epitope Y1214; and-   (ii) isolating an antibody from said mammal.

The skilled man would have access to a large number of assay techniqueswith which to develop assays to detect, measure the levels of, and/ormeasure a change in the activation state of the KDR/Flk-1 receptor,based on the knowledge of the importance of the Y1214 epitope. Examplesof such assay techniques include, fluorimetic assays, chromogenicassays, radiolabelled assays or chemiluminescence assays. Thus,according to a third aspect of the invention there is provided an assaymethod to detect, measure the levels of, and/or measure a change in theactivation state of the KDR/Flk-1 receptor utilising Y1214 as a marker,preferably to measure a change in the activation state of the KDR/Flk-1receptor.

For guidance for the design of biological assays the skilled man will beaware of a number of standard works. Examples of such standard worksinclude: Knight (1995) Fluorimetric Assays of Proteolytic Enzymes,Methods in Enzymology 248, 18-34; Chard (1978) An Introduction toRadioimmunoassay and related techniques, Elsevier/North-HollandBiomedical Press; and Basic Methods in Molecular Biology. First authorLeonard G Davis. 1986 Elsevier Science Publishing Co. Inc., Amsterdam.

According to a further feature of the third aspect of the inventionthere is provided an assay method for the measurement of a change in theactivation state of the KDR/Flk-1 receptor, comprising

-   (a) mixing a biological sample with an assay mixture comprising one    or more probes directed to the KDR/Flk-1 epitope, Y1214; and-   (b) measuring a signal which is proportional to the proportion of    the KDR/Flk-1 epitope, Y1214 which is in the phosphorylated or    un-phosphorylated state.

According to a further feature of the third aspect of the inventionthere is provided an assay method for the measurement of a change in theactivation state of the KDR/Flk-1 receptor, comprising

-   (a) mixing a biological sample with an assay mixture comprising one    or more probes directed to the KDR/Flk-1 epitope, Y1214; and-   (b) measuring the activation state of the KDR/Flk-1 receptor in the    biological sample.

According to a fourth aspect of the invention there is provided adiagnostic kit to detect, measure the levels of, and/or measure a changein the activation state of the KDR/Flk-1 receptor comprising reagentsfor preparing an assay mixture comprising a probe of the invention andinstructions for use thereof. Preferably said diagnostic kit is directedtowards measuring the activation state of the KDR/Flk-1 receptor.

According to a further feature of the fourth aspect of the inventionthere is provided a diagnostic kit to detect, measure the levels of,and/or measure a change in the activation state of the KDR/Flk-1receptor in a biological sample, for use with a method of the invention,the diagnostic kit comprising reagents for preparing an assay mixtureand instructions for use thereof.

According to a further feature of the fourth aspect of the inventionthere is provided a method of determining the effective dose of aninhibitor of the KDR/Flk-1 receptor, which comprises:

-   (a) dosing one or more humans or other mammals with a range of    KDR/Flk-1 receptor inhibitor concentrations, preferably one or more    humans;-   (b) isolating a biological sample from said humans or other mammals,    preferably a cell sample;-   (c) measuring a signal proportional to the activation state of the    KDR/Flk-1 receptor using an assay method of the invention; and-   (d) calculating the effective dose of the inhibitor from the    measured signal.

It would be clear to the skilled man that the KDR/Flk-1 receptorinhibitor is an inhibitor of the activation of the KDR/Flk-1 receptor bybinding of a ligand, such as VEGF, to the receptor. This could occur atthe VEGF binding site or at an allosteric site. Such inhibitors includeinhibitors of the KDR/Flk-1 receptor tyrosine kinase or agents whichblocks binding of ligands to the receptor, for example antibodiesdirected to the KDR/Flk-1 receptor. A preferred inhibitor isN-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(4-methylpiperidin-1-yl)methoxy]quinazolin-4-amine.

According to a further feature of the fourth aspect of the inventionthere is provided a method of determining the effective therapeutic doseof a KDR/Flk-1 receptor inhibitor which comprises:

-   (a) dosing one or more humans or other mammals with a range of    KDR/Flk-1 receptor inhibitor concentrations, preferably one or more    humans;-   (b) isolating a biological sample from said humans or other mammals,    preferably a cell sample;-   (c) measuring the activation state of the KDR/Flk-1 receptor using a    method of the invention; and-   (d) calculating the effective therapeutic dose of the inhibitor from    the measured activation state.

According to a the fifth aspect of the invention there is provided amethod of preparing a pharmaceutical composition of a KDR/Flk-1 receptorinhibitor which comprises:

-   (a) determining the effective dose of the inhibitor according to a    method of the invention; and-   (b) preparing a unit dose of inhibitor comprising an amount of    inhibitor within the effective dose range and a pharmaceutically    acceptable excipient.

According to a further feature of the fifth aspect of the inventionthere is provided a method of preparing a pharmaceutical compositionwhich comprises:

-   (a) determining the effective therapeutic dose of a KDR/Flk-1    receptor inhibitor by a method of the invention, and-   (b) mixing the inhibitor in an amount within the effective    therapeutic dose range with a pharmaceutically acceptable excipient.

According to a further feature of the fifth aspect of the inventionthere is provided a pharmaceutical composition prepared by one of theabove methods of preparing a pharmaceutical composition.

According to a sixth aspect of the invention there is provided a methodof determining whether a chemical compound is an in-vivo inhibitor ofthe KDR/Flk-1 receptor tyrosine kinase activity which comprises,measuring the degree of phosphorylation of Y1214 in a biological sampleobtained from a subject to whom said chemical compound has beenadministered.

According to a seventh aspect of the invention there is provided the useof the degree of phosphorylation of Y1214 as a surrogate marker ofKDR/Flk-1 receptor tyrosine kinase inhibitory activity of a chemicalcompound.

The skilled man will appreciate that this effective therapeutic dose maybe formulated in one or more preparations to be delivered to the patientover a defined period of time. The skilled man will also appreciate thatonce the effective dose has been determined further pharmaceuticalcompositions can be prepared which will have been indirectly prepared bythe above methods.

Within this specification the following terms are defined as follows:

-   “Activation state”—any change in the molecular conformation of the    KDR/Flk-1 receptor which leads to the direct or indirect modulation    of the activity of another protein or other proteins within the cell    membrane or any other part of a animal or human cell. Preferably the    change in conformation is induced directly or indirectly by the    phosphorylation of one or more amino acid residues within the    KDR/Flk-1 receptor.-   “Antibodies” include polyclonal antibodies, monoclonal antibodies,    and the various types of antibody constructs such as for example    F(ab′)₂, Fab and single chain Fv. Antibodies are defined to be    specifically binding if they bind Y1214 with a K_(c) of greater than    or equal to about 10⁷ M⁻¹. Affinity of binding can be determined    using conventional techniques, for example those described by    Scatchard et al., Ann N.Y. Acad. Sci., 51:660 (1949).-   “Assay Mixture”—comprises reagents suitable for the detection,    measurement of the levels of, and/or measurement of a change in the    activation state of the KDR/Flk-1 receptor; such reagents are    readily selectable by a person skilled in the art. For example, a    preferred assay mixture comprises a suitable buffer, one or more    labelled probes directed to the KDR/Flk-1 epitope, Y1214, and    optionally any other co-factors which may be required.-   “Biological sample” is a sample of material derived from any    component of an animal body. Such material includes, but is not    limited to, blood, urine, tissue sections, and tissue biopsies,    preferably tissue biopsies or blood. Tissue biopsies may be assayed    as sections or may be assayed as a cell suspension. Biological    samples may be derived from a variety of animal species, preferably    mammals, more preferably human, monkey, dog, guinea pig, rat or    mouse, further preferably rat, mouse or human, most preferably    human. The skilled man would appreciate that a biological sample may    be purified or partially purified before use. “Biological sample’    also includes cultured cell lines, and material derived therefrom. A    preferred biological sample is a needle biopsy from a tumour.-   “Effective Dose”—for the avoidance of doubt effective dose means the    effective therapeutic dose for the treatment of a disease wherein    inhibition of the activity of the KDR/Flk-1 receptor would be of    therapeutic benefit. The skilled man would be able to calculate the    effective therapeutic dose from the degree of inhibition of the    activity of the KDR/Flk-1 receptor in the biological sample.-   “KDR/Flk-1 epitope Y1214”—for the avoidance of doubt, references to    the KDR/Flk-1 epitope Y1214 relate to the epitope in both its    un-phosphorylated and phosphorylated form.-   “Modulation”—for the avoidance of doubt modulation can relate to    either an increase in activity or a decrease in activity.-   “Probe”—relates to any moiety which specifically binds to an amino    acid sequence, for example a sequence containing the Y1214 epitope    of the KDR/Flk-1 receptor.-   “Signal”—for the avoidance of doubt a signal could be derived from a    single probe or could be a number of signal-derived from two or more    probes. For example, a probe directed to the phosphorylated epitope    could be used in combination with a probe directed to the    un-phosphorylated epitope and the resulting 2 signals be used to    measure the proportion of receptor in the phosphorylated and    un-phosphorylated state.-   “Surrogate marker”—relates to any measurement of a biological or    biochemical activity which indirectly indicates the efficacy of a    chemical compound on the treatment of a disease state in an animal,    preferably a human.-   “Y1214”—relates to the tyrosine residue located at position 1214 in    the amino acid sequence of the mature KDR/Flk-1 receptor. The    sequence is set out in Terman et al 1991, Biochem Biophys Research    Commun 187(3), 1579-1586. In the Terman et al sequence the initiator    methionine is present, thus Y1214 is shown at position 1215 of the    sequence. This methionine would be absent in the transcribed    protein; thus the tyrosine residue relating to the present invention    would be present at position 1214. The sequence of KDR/Flk-1 can    also be found at SwissProt Accession No. P35968.

The invention will now be illustrated with reference to the followingnon-limiting examples and accompanying figure, wherein:

FIG. 1: shows a dose dependent decrease in phosporylation of the Y1214eptitope in response to increasing concentration of the KDR receptortyrosine kinase inhibitor4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-(3-piperidin-1-ylpropoxy)quinazoline;where lane O=untreated HUVEC cell lysate, lane V=VEGF-treated HUVEC celllysate and the remaining lanes show the effect of 0.007-5.0 uM compoundon VEGF-treated cells. The arrow shows the position of KDR on themembrane.

and the following abbreviations are used:

-   PBST—0.05% Tween-20 in PBS.-   BSA—bovine serum albumin;-   HUVECs—human unbilical vein endothelial cells-   PBS—phosphate-buffered saline; and-   OVA—ovalbumin

EXAMPLE 1 Preparation of Polyclonal Antibodies Directed to the KDR/Flk-1Epitope, Y1214

Conjugation of KDR/Flk-1 Peptide to OVA Using Maleimide

KDR/Flk-1 peptide “Ac-CDPKFHY(p)DNTAGIS-NH₂” (SEQ ID NO: 1) wasdissolved in PBS (0.1M pH=7.4) at a concentration of 1 mg/ml andfreeze-dried maleimide activated ovalbumin (ex Pierce & Warriner Cat.No. 77126) was dissolved in PBS at 1 ml/mg. 3 mls of peptide was thenmixed with 3 mls of maleimide-activated OVA and left to react for 4hours at room temperature in the dark, with continuous mixing on aroller. After 4 hours the reaction mixtures were dialysed against 10liters of stirred PBS overnight at 4° C. (10000 molecular weight cutoff). After dialysis the volumes of the reaction mixtures were adjustedto give final protein concentration of 1 mg/ml. 200 μl aliquots werethen stored at −20° C.

Immunisation of Rabbits with KDR/Flk-1 Peptide Conjugate

Three female New Zealand White rabbits were immunised with the ovalbuminconjugate of the KDR/Flk-1 peptide. Immunisations were carried out usingthe subcutaneous route, each dose being delivered to 4 sites on the backof the rabbit, in a total volume of 1 ml. Four doses were given at fourweekly intervals, with each dose containing 50 μgs of conjugate, asfollows.

-   Dose 1—delivered as an emulsion, made up using a 1:1 mixture of PBS    and Freunds Complete Adjuvant;-   Dose 2—delivered as an emulsion, made up using a 1:1 mixture of PBS    and Freunds Incomplete Adjuvant;-   Dose 3—delivered as an emulsion, made up using a 1:1 mixture of PBS    and Freunds Incomplete Adjuvant; and-   Dose 4—delivered in PBS alone.

Pre-bleeds were taken prior to the first dose and 10 days after eachsubsequent dosing. The blood was removed from the marginal ear vein ofthe animal, allowed to clot for several hours at room temperature andthen stored at 4° C. overnight. The samples were then spun in abench-top centrifuge for 15-30 minutes and the serum decanted from thepellet. The serum samples were then frozen and stored at −20° C.

Assay of Test Bleeds from Rabbits Immunised with Phosphorylated Peptide

Phosphorylated [Ac-CDPKFHY(p)DNTAGIS-NH₂] (SEQ ID NO: 1) Non[Ac-CDPKFHYDNTAGIS-NH₂] (SEQ ID NO: 2) Phosphorylated

Assay were performed on 96 well polystyrene plates. Plates were coatedwith either phosphorylated or non-phosphorylated peptide at 2 μg/ml inbicarbonate buffer (0.1M, pH 9.6) and incubated for 8 hrs at roomtemperature. Plates were then washed once with PBS/Tween 20(0.5%)(pH=7.4) and blocked with a 1% solution of dried, skimmed milkpowder in PBS at 120 μl/well overnight at 4° C.

Test bleeds were then assayed as follows. Plates were then washed oncewith PBS/Tween20 (0.5%)(pH7.4), samples added to wells at 100 μl/well;doubling dilutions, starting with neat serum, in duplicate and thenincubated for 3 hours room temperature. Plates were then washed threetimes with PBS/Tween20 (0.5%)(pH7.4) and a peroxidase labelled Goat antiRabbit antibody (Sigma Chemical Co, Poole, Dorset, UK Catalogue NumberA-0545) diluted to 1 in 4000 with PBS/Tween20 (0.5%)(pH7.4) was added at100 μl/well. Plates were then incubated for 3 hours at room temperature.Plates were then washed four times with PBS/Tween20 (0.5%)(pH7.4). Then,peroxidase substrate, 600 μg/ml ortho-phenylene-diamine+0.02% urea H₂O₂(in citrate phosphate pH 5) substrate was added at 100 μl/well to theplates and incubated at room temperature. The reaction was stopped after15 minutes by the addition of 0.1M citric acid at 50 μl/well.

Plates then read at 450 nm using a spectrophotometric plate reader.

Preparation of Phosphorylated KDR/Flk-1 Peptide and Non-PhosphorylatedKDR/Flk-1 Peptide Affinity Columns Using “Sulfolink™”

Sulfolink™ is a 6% cross-linked beaded agarose gel with immobilisediodoacetyl groups for immobilising sulfhydryl-containing peptides.(Source: Pierce Chemical Company, Rockford, Ill., USA).

10 mls of “Sulfolink” slurry was added to a disposable 5 ml (bed volume)polypropylene columns with additional reservoir fitted at roomtemperature and allowed to sediment for 2 hours. The column was thenwashed twice with 25 mls of 50 mM Tris+5 mM Na-EDTA pH 8.5. Peptideswere dissolved at 1 mg in 5 mls of Tris/EDTA (as above).

Affinity columns were prepared by adding a 5 ml solution of either thephosphorylated or non-phosphorylated peptide, the columns were thenmixed well using a long needle, capped and mixed on shaker for 15minutes. After 15 minutes columns were set upright and left to settlefor 30 minutes. Then columns were drained and 6 mls of 50 mM Cysteine inTris/EDTA run into each column. The columns were then capped, mixedthoroughly with needle, and mixed for 15 minutes. Column were then setupright and allowed to settle for 30 minutes. 40 mls of 1M NaCl was thenrun through each column, followed by 30 ml 0.05% Sodium Azide. Columnswere then stored at 4° C.

Purification of Antibodies Using Non-Phosphorylated Peptide andPhosphorylated Peptide Affinity Columns

Prior to column chromatography serum samples were purified byprecipitation using 50% ammonium sulphate. The precipitates wereresuspended in approximately 10 mls of PBS and dialysed against 10Litres of PBS overnight at 4° C. (10,000 molecular weight cut off). Theammonium sulphate-purified samples were then further purified bychromatography on a phosphorylated peptide affinity column followed bychromatography on a non-phosphorylated peptide column. Prior to useaffinity columns prepared as described above with phosphorylated ornon-phosphorylated peptide were brought to room temperature and washedwith 20 mls of PBS.

Column chromatography was performed as follows.

-   (i) Phosphorylated Peptide Affinity Column

10 mls of ammonium sulphate purified material was made up to 50 mls withPBS and added to the top of a phosphorylated peptide affinity column andallowed to run through, then the column was washed with 15 mls of PBS.The column was then eluted with approximately 10 mls of 100 mM GlycinepH 3.0 and 10×1 ml fractions were collected, with 100 μl of 1M Tris pH8.9 being added to collection vials from fraction 4-10. The elution fromthe column was checked by measuring the optical density of the fractionat 280 nm in a spectrophotometer. The column was then regenerated by theaddition of 10 ml 100 mM Glycine pH 2.5, followed by 20 mls PBS and thecolumn was stored at 4° C.

-   (ii) Non-Phosphorylated Peptide Affinity Column

Fractions 3-8 were pooled and added to the non-phosphorylated peptidecolumn, followed by 10 mls of PBS. All material coming through thecolumn was collected in 1 ml fractions and the optical density at 280 nmmeasured checked on a spectrophotometer. Fractions 2-7 were then pooledand dialysed against PBS, overnight at 4° C. After dialysis, the sampleswere stored at 4° C. for immediate use, or at −20° C. for storage.

Assay of Rabbit Fractions from Phosphorylated Peptide andNon-Phosphorylated Peptide Affinity Columns

96 well plates were coated for 4 hours at room temperature with 2 μg/mlof peptide in 0.1M bicarbonate buffer pH 9.6. The plates were thenwashed once with PBS/Tween20 (0.5%)(pH=7.5) and blocked with a 1%solution of dried, skimmed milk powder at 200 μl/well for 1 hour at roomtemperature. Plates were then washed once with PBS/Tween20 (0.5%)(pH7.5)and samples added to wells at 100 μl/well. Fractions were assayed indoubling dilutions, starting at 25 ug/ml of protein (by optical densityat 280 nm). Plates were then incubated at 4° C. overnight.

Next morning plates were washed three times with PBS/Tween20(0.5%)(pH7.5) and a goat anti-rabbit peroxidase antibody (Sigma ChemicalCo, Poole, Dorset, UK, Catalogue Number:A-0545) diluted to 1 in 2000with PBS/Tween20 (0.5%)(pH7.5) was added at 100 μl/well. Plates werethen incubated for 4 hours at 4° C. After 4 hours plates were washedfour times with PBS/Tween20 (0.5%)(pH7.5). Then, the peroxidasesubstrate ortho-phenylene diamine+urea H₂O₂ (in citrate phosphate bufferpH=5) at 100 ul/well was added. After 3 minutes the reaction was stoppedby addition of 0.1M Citric acid 100 ul/well. Plates were then read at450 nm on a spectrophotometric plate reader.

EXAMPLE 2 Treatment of Human Umbilical Vein Endothelial Cells (HUVECs)with Inhibitors of KDR/Flk-1 Receptor Tyrosine Kinase

HUVECs were obtained from TCS Cellworks, Buckingham, UK (Cataloguenumber ZHS 8965) and cultured in Large Vessel Endothelial Cell BasalMedium+recommended supplements obtained from TCS Cellworks (cataloguenumbers ZHM 2951 and ZHS 8965). Cells were used when close to confluencein 3×75 cm² tissue culture flasks. Prior to treatment with inhibitorcells were cultured overnight in Basal Medium without supplements.

HUVECS were treated with inhibitors as follows. Inhibitors were dilutedin Basal Medium, added to cultures at a final concentration of 1 μM. andthe cells incubated at 37° C. for 82 minutes. After 82 minutes 50 ng/mlVEGF was added to the cells and the cells incubated for a further 8minutes at 37° C. After this further incubation the culture medium wasremoved from the cells and the cells lysed with 2.5 ml sample buffer[Sample buffer: 4 parts NuPage sample buffer (Novex, NP0007, Invitrogen,Paisley, Scotland) to 1 part distilled water]. The lysate was collected,3 μl of β-mercaptoethanol added and heated to 95° C. for 10 minutes toensure complete reduction and denaturation. The lysates can be stored at−20° C. until analysed.

EXAMPLE 3 SDS PAGE of Lysates

Lysates were analysed using SDS polyacrylamide gel electrophoresis(PAGE) to separate the proteins within the lysates. Electrophoresis wasperformed using a 4-12% NuPage bis-tris gel (Invitrogen, Paisley,Scotland, UK, catalogue no. NP0321) and NUPAGE MOPS SDS Running Buffer(Invitrogen, Paisley, Scotland, UK, catalogue no. NP0001). After theelectrophoresis the protein were transferred from the gels tonitrocellulose membrane using Novex blotting modules as per themanufacturers instructions (Invitrogen, Paisley, Scotland, UK, catalogueno. LC2001). The membranes were then stained with Ponceau S (Sigma,Poole, Dorset, UK. Catalogue No. P-7170) and store air-dried, in sealedbags at 4° C.

EXAMPLE 4 Treatment of Nitrocellulose Membranes with Anti-KDR/Flk-1Antibodies

Nitrocellulose membranes were incubated for 1 hour in 5% solution ofdried, skimmed milk powder in PBST [Marvel/PBST] at room temperature.After 1 hour the membrane was incubated for between 2-3 hours at roomtemperature with an anti-KDR/Flk-1 antibody diluted 1:100 inMarvel/PBST. After this period the membrane was washed four times for atleast 5 minutes with PBST and then the membrane was incubated with anappropriate secondary antibody (e.g. NEB an anti-rabbit antibody boundto horse-radish peroxidase; used at a dilution of 1:2000) for 1 hour atroom temperature. The membrane was then washed for 30 minutes using atleast 4 changes of PBST, incubated with Lumiglo reagents (Cell SignalingTechnology Inc, Cummings Center, Beverly, Mass. USA) as directed. Themembranes were then exposed to X-ray film and the film developed toreveal the bands reactive to the anti-KDR/Flk-1 antibody. Lumiglo is aproprietory mixture of reagents containing a substrate which isconverted to a chemiluminescent compound by the action of horseradishperoxidase. The light from this is detected by photographic film, forexample Kodak Biomax ML.

1. An isolated antibody generated using a peptide consisting of SEQ IDNO: 1 or SEQ ID NO: 2 as an immunogen.
 2. The antibody of claim 1,wherein the antibody is a monoclonal antibody.
 3. The antibody of claim1, wherein the antibody is a polyclonal antibody.
 4. A compositioncomprising the antibody of claim 1 and a carrier.
 5. A kit for detectingthe activation of KDR/Flk-1 comprising the antibody of claim 1 andreagents for a detection assay.
 6. The antibody of claim 1, wherein theantibody is a F(ab′)₂, a Fab, or a single chain Fv.
 7. A method ofgenerating an antibody, comprising immunizing an animal with a peptideconsisting of SEQ ID NO: 1 or SEQ ID NO: 2, and isolating the antibodyfrom the animal.
 8. The method of claim 7, wherein the animal is amammal.
 9. An isolated antibody generated by the method of claim
 7. 10.An isolated antibody that binds to a peptide consisting of SEQ ID NO: 1or SEQ ID NO:
 2. 11. A method for detecting the presence of KDR/Flk-1comprising mixing the antibody of claim 1 with a biological sample todetect the presence of KDR/Flk-1.
 12. A method for measuring the amountof KDR/Flk-1 in a sample comprising mixing the antibody of claim 1 witha biological sample and measuring the amount of KDR/Flk-1 in the sample.13. The method of claim 12, wherein measuring the amount of KDR/Flk-1 inthe sample comprises performing an assay selected from the groupconsisting of a fluorimetric assay, a chromogenic assay, a radiolabelledassay, and a chemiluminescence assay.